This study was undertaken to decipher the interdependent roles of (i) methylation within E2 binding site I and II (E2BS-I/II) and replication origin (nt 7862) in the long control region (LCR), (ii) expression of viral oncogene E7, (iii) expression of the transcript (E7-E1∧E4) that encodes E2 repressor protein and (iv) viral load, in human papillomavirus 16 (HPV16) related cervical cancer (CaCx) pathogenesis. The results revealed over-representation (p<0.001) of methylation at nucleotide 58 of E2BS-I among E2-intact CaCx cases compared to E2-disrupted cases. Bisulphite sequencing of LCR revealed overrepresentation of methylation at nucleotide 58 or other CpGs in E2BS-I/II, among E2-intact cases than E2-disrupted cases and lack of methylation at replication origin in case of both. The viral transcript (E7-E1∧E4) that produces the repressor E2 was analyzed by APOT (amplification of papillomavirus oncogenic transcript)-coupled-quantitative-RT-PCR (of E7 and E4 genes) to distinguish episomal (pure or concomitant with integrated) from purely integrated viral genomes based on the ratio, E7 CT/E4 CT. Relative quantification based on comparative CT (theshold cycle) method revealed 75.087 folds higher E7 mRNA expression in episomal cases over purely integrated cases. Viral load and E2 gene copy numbers were negatively correlated with E7 CT (p = 0.007) and E2 CT (p<0.0001), respectively, each normalized with ACTB CT, among episomal cases only. The k-means clustering analysis considering E7 CT from APOT-coupled-quantitative-RT-PCR assay, in conjunction with viral load, revealed immense heterogeneity among the HPV16 positive CaCx cases portraying integrated viral genomes. The findings provide novel insights into HPV16 related CaCx pathogenesis and highlight that CaCx cases that harbour episomal HPV16 genomes with intact E2 are likely to be distinct biologically, from the purely integrated viral genomes in terms of host genes and/or pathways involved in cervical carcinogenesis.

The tumor suppressor p53 maintains an equilibrium between self-renewal and differentiation to sustain a limited repertoire of stem cells for proper development and maintenance of tissue homeostasis. Inactivation of p53 disrupts this balance and promotes pluripotency and somatic cell reprogramming. A few reports in recent years have indicated that prevalent TP53 oncogenic gain-of-function (GOF) mutations further boosts the stemness properties of cancer cells. In this review, we discuss the role of wild type p53 in regulating pluripotency of normal stem cells and various mechanisms that control the balance between self-renewal and differentiation in embryonic and adult stem cells. We also highlight how inactivating and GOF mutations in p53 stimulate stemness in cancer cells. Further, we have explored the various mechanisms of mutant p53-driven cancer stemness, particularly emphasizing on the non-coding RNA mediated epigenetic regulation. We have also analyzed the association of cancer stemness with other crucial gain-of-function properties of mutant p53 such as epithelial to mesenchymal transition phenotypes and chemoresistance to understand how activation of one affects the other. Given the critical role of cancer stem-like cells in tumor maintenance, cancer progression, and therapy resistance of mutant p53 tumors, targeting them might improve therapeutic efficacy in human cancers with TP53 mutations.

We tested the hypothesis that (i) synonymous variations within the coding regions, and (ii) variations within the non-coding regions of HPV, influence cervical cancer (CaCx) pathogenesis under the impact of intact HPV16 genomes. Whole genome sequence analysis of HPV16 isolates within 70 CaCx cases and 25 non-malignant samples revealed that synonymous variations were significantly higher within the E6 (p = 0.014), E5 (p = 0.001) and L2 (p = 0.0002) genes of HPV16 isolates within cases, compared to isolates within non-malignant samples. All of the 25 (100%) humanized codons identified within L2 ORF of the samples analyzed, were harbored by CaCx cases, while 8 out of 25 (32%) were harbored by HPV16 positive non-malignant samples (p = 3.87105E-07). L2 (mRNA and protein) expression was evident only among cases with episomal viral genomes and L2 mRNA expression correlated significantly with E2 gene copy numbers suggesting expression from all episomal genomes. Among such cases, Asian American (AA) isolates portrayed all of the humanized codons (100%; 4–6/sample) recorded within L2, which was significantly higher (p = 2.02E-7) compared to the European (E) isolates (22.8%; none or 1–2/sample). Additionally, majority of E variant isolates within cases (54/57; 94.7%) portrayed a variation (T4228C) within the short non-coding region (NCR2) between E5 and L2 genes, which portrays a weak promoter activity specific for L2 mRNA expression. This resulted in loss of 9 out of 14 miRNA binding sites (hsa-miR-548 family), despite the significant overexpression of miR548a-5p and miR548d-5p among such cases (28.64 and 36.25 folds, respectively), in comparison to HPV negative control samples. The findings exemplify the biological relevance of sequence variations in HPV16 genomes and highlight that episomal HPV16 in CaCx cases employ multiple mechanisms to sustain L2 expression, thereby justifying the potential role of L2 in such cancers, as opposed to those harboring viral integration.

In a novel attempt to understand the variations in DNA sequences underlying HLA class I alleles associated with HPV16-related CaCx, we determined the alleles by reconstructing SNP-based haplotypes from resequencing of the most polymorphic exons 2 and 3 of HLA-A, HLA-B and HLA-C. We also determined the impact of SNPs and transcriptional alterations of the genes on CaCx. A high density of SNPs was identified from resequencing. HLA expression was determined by real-time PCR. We identified that even a single associated HLA allele had many underlying SNP-based haplotypes. Out of the most frequent (≥5%) HLA class I alleles, HLA-B*40:06 and HLA-B*15:02 respectively imparted significant risk towards and protection from CaCx as well as HPV16 infection. Employing median-joining networks to detect clusters of sequence-variations for specific HLA alleles, we found the protective SNP-based signature, GAATTTA, in all SNP-based haplotypes of HLA-B*15:02 allele. The signature was derived from seven SNPs within HLA-B which were newly associated with the disease. Contrarily, similarly derived risk-signature, TTGCGCC, mapped only to 52% of SNP-based haplotypes of HLA-B*40:06 allele. This indicated that all SNP-based haplotypes underlying a particular associated HLA allele might or might not have a single signature of risk/protection. HLA-A, HLA-B and HLA-C expressions were downregulated among CaCx cases compared to asymptomatic infections and HPV-negative controls. HLA-A and HLA-B were repressed in both cases harbouring episomal and integrated HPV16, whereas HLA-C in only the latter. Novel genetic variations and differential downregulation-patterns of HLA class I have a significant bearing on HPV16-related CaCx pathogenesis.

Ovarian cancer ranks fifth in terms of cancer mortality in women due to lack of early diagnosis and poor clinical management. Characteristics like high cellular proliferation, EMT and metabolic alterations contribute to oncogenicity. Cancer, being a “metabolic disorder,” is governed by various key regulatory factors like metabolic enzymes, oncogenes, and tumor suppressors. Sirtuins (SIRT1‐SIRT7) belong to the group of NAD + deacetylase and ADP‐ribosylation enzymes that function as NAD + sensors and metabolic regulators. Among sirtuin orthologs, SIRT6 emerges as an important oncogenic player, although its possible mechanistic involvement in ovarian cancer advancement is still elusive. Our data indicated a higher expression of SIRT6 in ovarian cancer tissues compared with the non‐malignant ovarian tissue. Further, we observed that overexpression of SIRT6 enhances glycolysis and oxidative phosphorylation in ovarian cancer cells. The energy derived from these processes facilitates migration and invasion through invadopodia formation by reorganization of actin fibers. Mechanistically, SIRT6 has been shown to promote ERK1/2‐driven activatory phosphorylation of DRP1 at serine‐616, which has an obligatory role in inducing mitochondrial fission. These fragmented mitochondria facilitate cell movement important for metastases. siRNA‐mediated downregulation of SIRT6 was found to decrease cellular invasion through compromised mitochondrial fragmentation and subsequent reduction in stress fiber formation in ovarian cancer cells. Thus, the present report establishes the impact of SIRT6 in the regulation of morphological and functional aspects of mitochondria that modulates invasion in ovarian cancer cells.

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Introduction:Human papillomavirus (HPV) types 16/18 are reportedly most common in cervical cancer (CaCx) with geographical variation of genotypes. HPV16 predominates both in squamous cell carcinoma (SCC) and adenocarcinoma in India, contrary to reported global predominance of HPV18 in the latter. Our study was aimed to determine the occurrence of HPV16/18 among histopathological types of cervical intra-epithelial neoplasia (CIN) and invasive CaCx from North Bengal, India and to identify any major deviation from the known Indian scenario of distribution of HPV16/18 genotypes in cases of SCC and adenocarcinoma.Materials and Methods:This was a retrospective, cross-sectional, case-only type of study, in which 40 cases were histopathologically diagnosed as CIN/CaCx, on which polymerase chain reaction (PCR), deoxyribonucleic acid (DNA)-sequencing and bioinformatics by basic search local alignment tool were performed for HPV-genotyping.Statistical Analysis:The distribution of HPV genotypes among cases of SCC and adenocarcinoma was compared by Fisher's exact-test.Results:HPV was detected in 97.5% (39/40) cases. HPV16-infected cases (32/39; 82.05%) predominated over HPV18-infected ones (7/39; 17.95%). However, HPV18-only infection was significantly (P = 0.0045, one-sided Fisher's exact test) more among adenocarcinoma (3/4; 75%) than SCC (2/26; 7.69%) contrary to HPV16-only infection (SCC = 24/26, 92.31%; adenocarcinoma = 1/4; 25%) whereas both CIN3 cases were HPV16-positive.Conclusion:Predominance of HPV18 over HPV16 in cases of adenocarcinoma in this region was contrasting to that of earlier Indian studies suggesting research on HPV18 related cervical carcinogenesis. PCR and DNA-sequencing could prove to be highly effective tools in HPV detection and genotyping. The study reported HPV16/18 infection in almost 98% of the cases, the knowledge about which might prove useful in future population based studies on HPV genotyping and designing of appropriate HPV-vaccines for this region.

Homeobox gene families are associated with embryonic development and organogenesis. Pieces of evidence suggest that these Homeobox genes are also crucial in facilitating oncogenesis when mutated or overexpressed. Paired homeodomain transcription factor-2 (PITX2), one of the members of this family, is involved in oncogenic regulation apart from its different development regulatory functions. PITX2 has been earlier shown to induce ovarian cancer cell proliferation through the activation of different signaling cascades.Increased cancer cell proliferation requires a constant supply of nutrients for both adenosine triphosphate and biomass synthesis, which is facilitated by altered cancer cell metabolism that includes enhanced glucose uptake and increased glycolytic rate. This present study highlights the involvement of PITX2 in enhancing the cellular glycolysis pathway in ovarian cancer cells through protein kinase B-phosphorylation (phospho-AKT). PITX2 expression correlates positively with that of the glycolytic rate-determining enzyme, lactate dehydrogenase-A (LDHA), in both high-grade serous ovarian cancer tissues and common ovarian cancer cell lines. Interestingly, transient localization of enzymatically active LDHA in the nucleus was observed in PITX2-overexpressed ovarian cancer cells. This nuclear LDHA produces higher concentrations of the glycolytic end product, lactate, which accumulates in the nuclear compartment resulting in decreased histone deacetylase (HDAC1/2) expression and increased histone acetylation at H3/H4. However, the mechanistic details of lactate-HDAC interaction are still elusive in the earlier reports. Our in silico studies elaborated on the interaction dynamics of lactate in the HDAC catalytic core through ligand-binding studies and